SUMMARY In the hematopoietic system, aging has been shown to result in dramatic changes in the clonal composition of the hematopoietic stem cell (HSC) pool, a shift their developmental potential, and a degradation of their overall activity. The broad goal of this Project is to understand the mechanisms that underlie these changes with a view to enhancing their activity and reverting their behavior to a more youthful state. Through deep epigenetic profiling, we showed that aged murine HSCs exhibit marked changes in the distribution of several epigenetic markers including DNA methylation and a set of histone marks. In humans, clonal evolution in the stem cell compartment is associated with mutations in epigenetic modifiers, particularly those involved in regulating DNA methylation. These data strongly suggest that changes in epigenetic regulation are leading to some of the deficiencies in HSC function that occur with age. We hypothesize that changes in the adaptive landscape with age promote the expansion and selection of HSCs with specific characteristics, acquired either through epigenetic drift and/or through mutations in epigenetic regulators. To address these questions, we will (1) Examine the epigenetic and gene expression changes characteristic of aged HSCs. In collaboration with Projects 1 and 2 and the Bioinformatics Core, we will comprehensively map age-driven chromatin remodeling, profiling in parallel MuSCs, NSCs, and HSCs. This will allow us to identify both core? and cell type-specific aspects of chromatin and epigenetic regulation. We will also examine nucleosome occupancy and variation in repetitive elements with age in HSCs. (2) We will examine the link between mutations in epigenetic regulators and age-associated epigenetic changes, and the the ability of the environment to promote these change in mutant HSCs. We will examine epigenetic changes in aged versus Dnmt3a-KO HSCs, test the behavior of 3aKO HSCs in an aged vs. young environment, or in an inflammatory milieu. (3) We will test the impact of rejuvenation interventions on age-associated phenotypes in the hematopoietic system. We hypothesize that specific interventions may slow acquisition of age-associated HSC phenotypes. In collaboration with Projects 1 and 2 and Core B, we will examine heterochronic parabiosis, exercise, rapamycin and cyclic fasting regimens to attempt to induce rejuvenating effects on stem cells. In addition, we will specifically explore the use of DNA methylation to induce more youthful functional and molecular phenotypes. We will globally express DNMT3A using a Dox-inducible system. In addition, we will use a novel CRISPR-Cas9 system to induce DNA methylation at specific sites in order to test their importance for the HSC aging phenotype. Together, these experiments will reveal the central role of epigenetic changes associated with aging, and the impact on hematopoietic phenotypes. These studies will lay the groundwork for future attempts to modulate the local environment for stem cells to promote healthy aging.